Culturing the epiblast cells of the pig blastocyst

Summary Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.     

Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes

Type I restriction enzymes comprise three subunits encoded by genes designated hsdR, hsdM, and hsdS; S confers sequence specificity. Three families of enzymes are known and within families, but not between, hsdM and hsdR are conserved. Consequently, interfamily comparisons of M and R sequences focus on regions of putative functional significance, while both inter- and intrafamily comparisons address the origin, nature and role of diversity of type I restriction systems. We have determined the sequence of the hsdR gene for EcoA, thus making available sequences of all three hsd genes of one representative from each family. The predicted R polypeptide sequences share conserved regions with one superfamily of putative helicases, so-called ‘DEAD box’ proteins; these conserved sequences may be associated with the ATP-dependent translocation of DNA that precedes restriction. We also present hsdM and hsdR sequences for EcoE, a member of the same family as EcoA. The sequences of the M and R genes of EcoA and EcoE are at least as divergent as typical genes from Escherichia coli and Salmonella, perhaps as the result of selection favouring diversity of restriction specificities combined with lateral transfer among different species.     

Mutants in dolichol synthesis: conversion of polyprenol to dolichol appears to be a rate-limiting step in dolichol synthesis

Chinese hamster ovary (CHO) cells of the Lec9 recessive complementationgroup display a distinctive profile of resistance to a varietyof toxic lectins. In addition, they accumulate cis--unsaturatedpolyprenol and use mainly polyprenol rather than dolichol tosynthesize the glycosylated lipids used in asparagine-linkedglycosylation of proteins. The primary defect in these cellsis thought to result from a deficiency in polyprenol reductaseactivity. Three new mutants were isolated and determined tohave qualitatively, although not quantitatively, similar lectinresistance profiles to Lec9 cells. Two of these mutants (Abr^Rand Ric^R) also contained polyprenol rather than dolichol. Thelectin resistance profile of an independent mutant which accumulatespolyprenol, F2A8, was also found to be qualitatively similarto the Lec9 pattern. The relationship among these mutants wasanalysed in more detail by construction of cell—cell hybrids.Lectin resistance profiles of the hybrids demonstrated thatAbr^R, Ric^R and F2A8 fell into the Lec9 complementation group.Analysis of prenols in the hybrids also showed that F2A8 wasa member of the Lec9 group. Surprisingly, a significant fractionof the prenols found in Lec9 Parent hybrids was polyprenol(up to 30% of the neutral fraction), whereas the prenols foundin Parent Parent hybrids were nearly exclusively dolichol(97% of the neutral lipid fraction). Therefore, reduction ofpolyprenol to dolichol appears to be a rate-limiting step inthe synthesis of dolichol since hybrids with differing numbersof wild-type alleles can be biochemically distinguished. CHO cells dolichol lectins mutants polyprenol reductase     

Hatching plasticity in a Southeast Asian tree frog mitigates submergence-induced mortality

Environmentally cued hatching has been well-documented in amphibians in response to a wide range of abiotic and biotic factors. The hatching of terrestrial amphibian eggs in response to flooding may be basal within the group, but amphibian lineages in tropical Asia and sub-Saharan Africa have not received as much attention as their Neotropical counterparts. We investigated submergence-induced hatching in Feihyla hansenae, a Rhacophorid tree frog with terrestrial eggs. We quantified natural rates of clutch submergence at our study site in Thailand. Using submergence experiments, we found that embryos are capable of hatching early to escape flooding, and that failure to hatch results in mortality. Among the embryos that were able to hatch early, only the earliest, youngest hatchlings experienced a trade-off in body size that persisted for 6 days, while later, older hatchlings were not significantly smaller than spontaneous hatchlings under control conditions. By incorporating our natural and experimental data into Monte Carlo methods to simulate and compare survival probabilities with and without hatching plasticity, we found an overall 3.1% increase in submergence survival due to hatching plasticity. Our findings support the idea that flooding-induced hatching is widespread across amphibians with terrestrial eggs and highlight the importance of researching understudied tropical regions. As climate change is projected to affect rainfall patterns, the ability of embryos to escape abiotic egg-stage threats may be an indicator of species' ability to flexibly navigate a changing environment.     

Morphology of the Bony Labyrinth Supports the Affinities of Paradolichopithecus with the Papionina

International Journal of Primatology - Discoveries in recent decades indicate that the large papionin monkeys Paradolipopithecus and Procynocephalus are key members of the Late Pliocene –...     

Characterization of 24 porcine (dA-dC)n-(dT-dG)n microsatellites: genotyping of unrelated animals from four breeds and linkage studies

Twenty-four PCR primer pairs were designed for the detection of porcine microsatellites. Polymorphism was investigated in 76 unrelated animals from four different breeds: Duroc, Landrace, Hampshire, and Yorkshire. Compared with human microsatellites, a general lower heterozygosity was detected; however, for each microsatellite a significant variation between breeds in number of alleles and heterozygosity was seen. Mean heterozygosity was found to be significantly higher (P<0.01%) in the Yorkshire breed than in the other three breeds. Linkage analyses with the CEPH linkage packet were performed in a backcross family comprising 45 animals, of which 43 had informative meioses. Ten of the microsatellites could be assigned to six different linkage groups, demonstrating that linkage mapping with microsatellites can be carried out with great efficiency in a relatively small number of animals. Four of the linkage groups represent Chromosomes (Chrs) 4, 6, 7, and 8 respectively, while two linkage groups are unassigned.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers listed in Table 1.     

Chromosomal localization of uroplakin genes of cattle and mice

The asymmetric unit membrane (AUM) of the apical surface of mammalian urinary bladder epithelium contains several major integral membrane proteins, including uroplakins IA and IB (both 27 kDa), II (15 kDa), and III (47 kDa). These proteins are synthesized only in terminally differentiated bladder epithelial cells. They are encoded by separate genes and, except for uroplakins IA and IB, appear to be unrelated in their amino acid sequences. The genes encoding these uroplakins were mapped to chromosomes of cattle through their segregation in a panel of bovine x rodent somatic cell hybrids. Genes for uroplakins IA, IB, and II were mapped to bovine (BTA) Chromosomes (Chrs) 18 (UPK1A), 1 (UPK1B), and 15 (UPK2), respectively. Two bovine genomic DNA sequences reactive with a uroplakin III cDNA probe were identified and mapped to BTA 6 (UPK3A) and 5 (UPK3B). We have also mapped genes for uroplakins 1A and II in mice, to the proximal regions of mouse Chr 7 (Upk1a) and 9 (Upk2), respectively, by analyzing the inheritance of restriction fragment length variants in recombinant inbred mouse strains. These assignments are consistent with linkage relationships known to be conserved between cattle and mice. The mouse genes for uroplakins IB and III were not mapped because the mouse genomic DNA fragments reactive with each probe were invariant among the inbred strains tested. Although the stoichiometry of AUM proteins is nearly constant, the fact that the uroplakin genes are unlinked indicates that their expression must be independently regulated. Our results also suggest likely positions for two human uroplakin genes and should facilitate further analysis of their possible involvement in disease.     

L'intoxication alloxanique chez la femelle gravide de cobaye

Sommaire Chez la femelle de cobaye gravide, une injection intracardiaque de 200 mg/kg d'alloxane provoque en 6–8 hs, une pycnose généralisée des cellules B des îlots endocriniens du pancréas; 18–20 hs après l'injection, les acini exocrines en bordure des îlots devenus nécrotiques se différencient et prolifèrent des cordons de cellules endocriniennes. Cette régénération intense et rapide est pratiquement achevée 42 hs après l'injection.L'effet, sur le pancréas foetal, de l'alloxane injectée à la femelle gravide, varie suivant le stade de développement atteint par le tissu endocrine au moment de l'injection. Pendant la phase de différenciation, aux dépens de l'épithélium des canaux, des premières cellules A (emb. de 13 mm, 25e jour) et B (emb. de 27 mm, 34e jour), puis de prolifération de cordons aboutissant à la formation des îlots primitifs dits îlots à manteau (foetus de 58 mm, 43e jour), les cellules B sont totalement réfractaires à l'action de l'alloxane. Chez les foetus ayant atteint 75 mm (50–51e jour), les premiers îlots à architecture définitive ont fait leur apparition par remaniement secondaire des îlots à manteau, processus qui continuera jusqu'à la naissance. Dès que ce stade est atteint, l'alloxane injectée à la mère provoque la pycnose généralisée des cellules B dans les îlots définitifs alors que les îlots à manteau voisins restent intacts. Il semble donc que les cellules B doivent atteindre un certain degré de maturité pour être sensibles à l'alloxane.     

GLOBAL POPULATION STRUCTURE AND NATURAL HISTORY OF THE GREEN TURTLE (CHELONIA MYDAS) IN TERMS OF MATRIARCHAL PHYLOGENY

To address aspects of the evolution and natural history of green turtles, we assayed mitochondrial (mt) DNA genotypes from 226 specimens representing 15 major rookeries around the world. Phylogenetic analyses of these data revealed (1) a comparatively low level of mtDNA variability and a slow mtDNA evolutionary rate (relative to estimates for many other vertebrates); (2) a fundamental phylogenetic split distinguishing all green turtles in the Atlantic-Mediterranean from those in the Indian-Pacific Oceans; (3) no evidence for matrilineal distinctiveness of a commonly recognized taxonomic form in the East Pacific (the black turtle C.m. agassizi or C. agassizi); (4) in opposition to published hypotheses, a recent origin for the Ascension Island rookery, and its close genetic relationship to a geographically proximate rookery in Brazil; and (5) a geographic population substructure within each ocean basin (typically involving fixed or nearly fixed genotypic differences between nesting populations) that suggests a strong propensity for natal homing by females. Overall, the global matriarchal phylogeny of Chelonia mydas appears to have been shaped by both geography (ocean basin separations) and behavior (natal homing on regional or rookery-specific scales). The shallow evolutionary population structure within ocean basins likely results from demographic turnover (extinction and colonization) of rookeries over time frames that are short by evolutionary standards but long by ecological standards.